The introduction of new traits by gene transfer has become routine with respect to many dicotyledonous plant species. In contrast, monocotyledonous plant species, particularly cereals, have proven to be recalcitrant to genetic engineering because they do not belong to the natural host range of Agrobacterium. Therefore, unlike dicots, monocots are generally not susceptible to gene transfer by Agrobacterium-mediated transformation. However, the application of such methods as electroporation, PEG-mediated transformation of protoplasts, and particle bombardment, have shown promise for the stable transformation of cereals. Moreover, successful transformation of monocots using hypervirulent Agrobacterium strains has been recently reported.
However, the step of the introduction of foreign DNA into plant cells is only part of the equation for obtaining plants that possess new traits. It is also necessary that the transformed cells be successfully regenerated to form viable plants. Despite advances in methods for transforming monocots with foreign DNA, regeneration of fertile plants from transformed somatic cells or tissues remains a challenge, particularly in barley and wheat. One problem is that, in monocotyledonous plants, only a few somatic tissues are totipotent, i.e. capable of being regenerated to form green, fertile plants. Plants can be regenerated from totipotent cells in tissue culture by either embryogenesis or organogenesis.
i) Embryogenesis
TABLE 1Comparison of zygotic and somatic embryogenesis(based on Dodeman et al., 1997)StepsZygotic embryogenesisSomatic embryogenesisOriginZygoteSomatic cellIn the ovuleIsolated or notHaploid cells (e.g.microspore)InitiationFecundation (exceptHormonal inductionapomixis)Low cell frequencyEvery zygoteDedifferentiationConstitutional polarityPolarity?Asymmetric divisionAsymmetric division?(under genetic control)ConstructionEmbryo/suspensorSimilar conditions, but withof an embryoEmbryo axis in placevariations:(under genetic control)Absence of the suspensorReorganization in proembryoclustersAdventitious embryogenesisMeristemTightly geneticallyInteraction between geneticformationcontrolled root meristemand hormonal controlsshoot meristemNumerous abnormalitiesMaturationStorage proteinAbsence of maturation andDehydrationendospermDormancy (genetic control,External induction factorsABA)(amino acids, sugars, ABA,Plant maternal tissue-dehydration)embryo interaction
Embryogenesis is the process of embryo initiation and development, and may be classified as either zygotic or somatic, according to the type of cell from which embryogenesis arises. Features of zygotic and somatic embryogenesis are summarized in Table 1 (based on Dodeman et al., 1997).
Zygotic embryogenesis relates to embryogenesis arising from the zygote or fertilized egg which originates in the ovule, and is intrinsically embryogenic. Proliferation of the zygote leads to the formation of a zygotic embryo within which organs and tissues begin development. Within the embryo, distinctive regions, designated as the shoot and root apical meristems, promote the development of shoot and root systems respectively. Activity of the meristems contributes to the continuing expansion and formation of the plant. During the maturation stage, storage protein accumulates and dehydration enhances germination. In zygotic embryogenesis, the cells are restricted to specific fates since the developmental stages (i.e. globular, heart, torpedo, cotyledonary) are strictly genetically controlled.
In comparison, somatic embryogenesis relates to embryogenesis arising from somatic cells (i.e. vegetative or non-gametic cells), namely from isolated somatic explants or microspores. Since somatic cells are not naturally embryogenic, such cells must be induced to become embryogenic. Conversion from somatic to embryogenic cells may be achieved by external stimuli such as auxin, cytokinin, pH shifts, growth regulators, and heavy metal ions (Yeung, 1995; Dodeman et al., 1997).
Successful formation of somatic embryos is largely dependent upon the explant tissue of choice (Merkle et al., 1995). Very young zygotic embryos form somatic embryos in response to cytokinin, whereas more mature zygotic embryos no longer respond to cytokinins alone and require auxin to form somatic embryos. Meristematic cells from grasses and other monocots behave similarly. In more differentiated tissue, auxin and cytokinin induce formation of calli from which somatic embryos may be produced.
Regardless of the explant source, the obtained somatic embryo may then follow a developmental pattern similar to that of a zygotic embryo. However, unlike zygotic embryogenesis in which the developmental fate of cells is programmed, somatic embryogenesis differs in that variations and abnormalities may arise during the stages of embryo construction and meristem formation. Variations observed in vitro include the absence of the suspensor; reorganization of cells in proembryo clusters; occurrence of adventitious embryogenesis; and other abnormalities due to interaction between genetic and external hormonal controls. In zygotic embryos, activity of the meristems is crucial towards the development of the plant, whereas in somatic embryos, little is known about meristem differentiation. In comparison to zygotic embryos, maturation and storage reserves are absent in somatic embryos. Notably, somatic embryos tend to germinate precociously, with abnormalities observed such as failure or uncoordination of shoot or root formation, multiple cotyledons, or precocious or abnormal shoot formation (Wetherell, 1979). Such abnormalities are not considered problematic since in general, plantlets with normal shoots and roots eventually form (Wetherell, 1979). The fate of the cells in somatic embryos is thus not as fixed as that of cells of zygotic embryos, which follow a determined and highly regulated pathway. However, somatic embryos are beneficial for their abilities to form a complete plant despite the natural mutations that may occur in vitro, and to germinate precociously, contributing to rapid regeneration of plants.
ii) Organogenesis
As an alternative to embryogenesis, plants may also be regenerated from totipotent cells in tissue culture by organogenesis, whereby new organs such as shoots and roots, rather than whole embryos as in embryogenesis, form directly from cultured cells. The process can occur directly on the explant or indirectly via calli formation. A significant feature of organogenesis is the development of a meristem or shoot/root primodium. Only one meristem is formed in organogenesis, while two meristems (one for a shoot and the other for a root) are produced in embryogenesis. However, it is not uncommon to encounter a zygotic embryo with only one meristem (a shoot primordium) upon dissection of an immature barley embryo.
At the physiological, biochemical and structural levels, embryogenesis and organogenesis have certain common features; thus, in morphogenetic in vitro studies, it may be difficult to confirm whether somatic embryogenesis or true organogenesis has occurred (Thorpe, 1993).
Due to variations which naturally occur with somatic embryos and factors such as the explant source, media, or tissue culture technique, cells may have different developmental fates, such that some cells produce embryos (i.e. embryogenesis), while others form shoot or root primordia (i.e. organogenesis).
Although both organogenesis and embryogenesis lead to regeneration of fertile plants, embryogenesis has certain advantages. First, somatic embryos, like zygotic embryos, naturally proceed through the developmental process to form a complete plant, with little intervention. In contrast, during organogenesis, separate shoot growth and rooting steps are usually required in order to obtain complete plantlets. Second, when cultured under appropriate conditions, rather than proceeding to the next developmental stage, a somatic embryo may instead give rise to new somatic embryos. This process has been described as secondary, recurrent, or repetitive embryogenesis. Because somatic embryos can be perpetuated via repetitive embryogenesis, they are attractive candidates for the mass production of clonal plantlets.
In monocot tissue culture, the most commonly used regenerable embryogenic tissues are embryogenic suspension cells and embryogenic callus cultures. Suspension cultures are substantially homogeneous suspensions of microcalli in liquid medium. Callus cultures are grown on solid media, develop larger contiguous masses of calli, and are more heterogeneous with respect to the embryogenic quality of the calli. Both suspension cells and calli have limitations as target tissues for transformation with foreign DNA. The preparation of suspension cells involves a lengthy in vitro culture period, and the cells exhibit a significant reduction of morphogenetic competence over time. Additionally, plants regenerated from suspension cells manifest substantial undesirable somaclonal variation, such as infertility or albinism.
Because the time needed for establishment of culture and plant regeneration is shorter than with suspension cells, embryogenic callus has been considered as possibly a preferable target tissue, but problems remain. Somaclonal variation persists, and most cells lose their ability to regenerate when they reach the callus stage (Jähne et al., 1995). Despite efforts to improve callus culture, only a few monocot genotypes can be successfully regenerated from calli. In North America, the small number of genotypes which have been used successfully to regenerate fertile plants from calli include the barley genotype Golden Promise, the winter barley genotype Igri, and the wheat genotypes Fielder, Bobwhite, and Chinese Spring. Moreover, cereal calli remain embryogenic only briefly, further compounding the difficulties experienced in tissue culture.
In view of the limitations of suspension cells and callus cultures, efforts have been directed towards using primary explants such as immature embryos and inflorescences as target tissues for obtaining stably-transformed plants. However, the tissue culture techniques which have been applied to primary explants result in indirect somatic embryogenesis, wherein the intermediate cell generations between the original explant and the formation of somatic embryos are manifested as calli. Hence, indirect somatic embryogenesis from primary explants does not entirely resolve the problems associated with regeneration of plants from callus cultures.
Typically, indirect somatic embryogenesis in tissue culture involves two distinct steps, induction and regeneration. During induction, the tissue of interest is cultured on an induction medium which encourages un-differentiation of cells, and induction of fast-growing embryogenic calli. The callus stage is characterized by rapid, anarchic cell division. Tissues are cultured on the induction medium for a fixed, predetermined period, which is of sufficient duration for the production of fast growing embryogenic calli. This period typically ranges from one to four weeks (Nehra et al., 1994; Becker et al., 1994). If necessary, the tissue may be subcultured on the same medium for an additional period of time (Cho et al, 1998).
The hormone content of the media is of greatest significance. The three major classes of plant growth regulators used in tissue culture are auxins, cytokinins, and polyamines. Auxins are involved in many aspects of cell biology and tissue development. The most common are the naturally-occurring auxins indole-3-acetic acid (IAA), indole butyric acid (IBA), phenylacetic acid (PAA), and the synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba (2-methoxy-3,6-dichlorobenzoic acid) and picloram (4-amino-3,5,6-trichloropiconilic acid). Like auxins, cytokinins are involved in many aspects of cell biology and tissue development, especially cell division. The naturally-occurring cytokinins benzyl amino purine (BAP), benzyladenine (BA) and zeatin, and the synthetic cytokinin kinetin are the most commonly used in tissue culture. Polyamines, which include spermine, spermidine and putrescine, are less well known than other plant growth regulators. Although their precise physiological role still remains to be determined, polyamines appear to influence cell division and embryogenesis in carrot cell culture. They also bind to nucleic acids, phospholipids and proteins to further stabilize these molecules.
A typical callus induction medium for barley and wheat is Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), which itself is hormone free, supplemented with 30 g/L maltose, 1.0 mg/L thiamine-HCl, 0.25 g/L myo-inositol, 1.0 g/L casein hydrolysate, 0.69 g/L proline, and 11.3 μM of dicamba (Wan and Lemaux, 1994). Other variants of basal MS medium are also known. For instance, Cho et al. (1998) describe an induction medium containing 4.5–11.3 μM 2,4-D, and 2.2 μM BAP. Nehra et al. (1994) and Bregitzer et al. (1998) teach induction media in which 9-13.6 μM 2,4-D is the only hormone component. Ritala et al. (1994) provide an induction medium in which the only plant hormone is 1.8 μM BAP. Barro et al. (1998) report that, depending on the conditions and tissues, the presence of 16.6 μM picloram can result in higher transformation efficiency than the presence of 2,4-D alone. Similarly U.S. Pat. No. 5,631,152 to Fry et al. teaches an induction medium containing 9.1 μM picloram and 2.2 μM 2,4-D.
Another basal medium commonly used for induction of callus culture is hormone free L3 medium supplemented with 30 g/L maltose and 9 μM of 2,4-D (Barcelo et al., 1993). The medium commonly used for induction of barley microspores culture is quite similar, and contains FHG basal medium supplemented with 63 g/L maltose, 730 mg/L glutamine, 100 mg/L myo-inositol, 0.4 mg/L thiamine-HCl, 4.4 μM BAP and 73.4 μM PAA (Yao et al., 1997).
In the second step, the calli are cultured on a regeneration medium such as MS, FGH, or L3. The regeneration medium is usually hormone free, though it may be supplemented with a very small amount of cytokinin and auxin, in the order of less than 4.5 μM. Termination of the auxin-mediated hormonal control allows embryogenesis to commence. As they mature, developing embryos produce shoots and regenerated plantlets. If necessary, the mass of cells with green shoots is excised and placed on a rooting medium. Rooting media typically do not contain plant hormones, although some may contain up to about 2 μM of auxin. The plantlets are then transferred to soil.
Although the two-step induction and regeneration approach to somatic embryogenesis has been applied to monocots, it has a number of significant disadvantages. First, since the induction step involves proliferation of calli, somaclonal variation remains a concern. Second, induction and regeneration are slow. Since the culture steps proceed according to a pre-determined time line, there is no opportunity to proceed more rapidly should the tissue reach the next developmental stage more quickly than anticipated. Generally, induction of calli and regeneration of green, fertile plants by indirect somatic embryogenesis takes at least three months.
In contrast to indirect somatic embryogenesis, a tissue culture process for direct somatic embryogenesis in monocotyledonous plants would advantageously avoid the callus step, thereby minimizing somaclonal variation. Moreover, such a process would also desirably eliminate the constraints of a pre-determined tissue culture schedule, thereby enabling plant regeneration to proceed as quickly as is biologically feasible. Direct somatic embryogenesis has been reported in dicots such as clover, carrot, and tobacco. For instance, Maheswaran and Williams (1984, 1985) disclose direct somatic embryogenesis of immature embryos of Trifolium repens (white clover), Trifolium pratense (red clover), and Medicago sativa (alfalfa) cultured on a basal nutrient medium (EC6) supplemented with 0.22 μM of the cytokinin BAP.
Despite reports of direct somatic embryogenesis in dicots, to the applicants' knowledge, direct somatic embryogenesis has not been accomplished in monocots. The patent literature discloses a number of methods for somatic embryogenesis in monocotyledonous plant tissues, but these involve a step of inducing calli, and therefore constitute indirect, rather than direct somatic embryogenesis. For instance, U.S. Pat. No. 5,631,152 to Fry et al. teaches indirect somatic embryogenesis in Triticum aestivum. U.S. Pat. Nos. 5,641,664 and 5,712,135 to D'Halluin et al. and U.S. Pat. No. 5,792,936 to Dudits et al., teach regeneration of corn plants from calli cells. U.S. Pat. No. 5,589,617 to Nehra et al. teaches a method for regenerating plants from wheat or barley embryos, which involves the induction of a callus stage. U.S. Pat. No. 5,610,042 to Chang et al. teaches a method for producing stably transformed fertile wheat plants involving a step of inducing formation of calli from immature wheat embryos. U.S. Pat. No. 5,874,265 to Adams et al. teaches production of stable, genetically transformed cereal plants, particularly wheat, barley, or oats, in which regeneration of plants from the transformed cells involves induction of calli. U.S. Pat. No. 4,666,844 to Cheng provides a process for regenerating cereal plants such as barley, corn, wheat, rice, and sorghum, in which tissues are first cultured under conditions sufficient to ensure calli formation. U.S. Pat. No. 5,981,842 to Wu et al. teaches the regeneration of transgenic rice (Oryza sativa), from calli induced from immature embryos. U.S. Pat. No. 5,409,828 to Frenkel et al. describes somatic embryogenesis in Asparagus officinalis, which is a monocot. But again, the first step is the induction of calli. Canadian Patent No. 1,292,959 to Stuart et al. describes somatic embryogenesis of corn and rice, again involving a callus step. International Publication No. WO 99/04618 to Rikiishi et al. discloses a method for producing transformed barley cells, which includes the step of culturing the barley cells in a calli induction medium.
Jähne et al. (1994) report some success in obtaining direct embryogenesis in barley microspores. However, microspores are germ cells, rather than somatic cells. Like other isolated germ cells, microspores are delicate relative to somatic cells, and they are very susceptible to damage by particle bombardment. Jähne et al's process is therefore of limited utility for introducing foreign genes into monocots, given that particle bombardment is the preferred transformation technique in monocots. Similarly, U.S. Pat. Nos. 5,322,789 and 5,445,961 to Genovesi et al. describe culturing corn microspores to obtain embryoids or calli.
Hence, there is a need for a tissue culture process suitable for effecting direct somatic embryogenesis in monocotyledonous plant cells or tissues and rapid regeneration of fertile plants. Ideally, in order to expedite the recovery of fertile plants, such a process would not impose predetermined time limits on the various tissue culture steps. Such a process would advantageously provide for recurrent or secondary embryogenesis from the developing embryos. In addition, such a process would also promote organogenesis in developing embryos. A direct somatic embryogenesis method for monocots would also desirably provide for the ready introduction of foreign genes into the plant.